Project Abstract: Brain metabolism must accommodate a wide dynamic range of energy demands from time to time and from cell type to cell type. Although the metabolic response to increased brain activity is the basis of well-known functional MRI signals, the nature of this metabolic response is still very controversial. It has been demonstrated that metabolism plays a key regulatory role in several neurological conditions, including epilepsy and neurodegenerative diseases. However, little is known about the mechanisms coupling neuronal excitability to metabolism, though the main metabolic pathways have been well established for decades now. The study of real-time dynamics of brain metabolism has been hampered by the insufficient spatial and temporal resolution of the methods, but such limitations can now be overcome by the use of genetically- encoded fluorescent biosensors. I will use a combination of sensors to determine glucose consumption as well as the NADH/NAD+ and ATP/ADP ratios, respectively. These sensors can give calibrated quantitative readouts within intact brain tissue, by using two-photon microscopy with either ratiometric or fluorescence lifetime imaging (FLIM). The Ca2+ indicator RCaMP1h and electrophysiologic recordings will provide me with a readout of neuronal activity. Hippocampal CA3 neurons can be excited synaptically (which also engages astrocytes) or antidromically. The proposed research combines the use of fluorescent glucose, ATP, NADH and Ca2+ biosensors with advanced electrophysiological and optical 2-photon imaging techniques to study the energy metabolism of neurons and astrocytes in brain slices. Aim 1 is to characterize the dynamics of glucose and NADH and ATP levels in the astrocytes and neurons (both glutamatergic and GABAergic) in hippocampal slices during resting and active states. This work will also explore the glycolytic contribution to the neuronal activity-induced acceleration of energy metabolism. Aim 2 is to determine the mechanisms linking the neuronal activity to an increased metabolism, both the triggering stimuli (Na+, K+ and Ca2+ fluxes) and subsequent signaling (such as ionotropic or G-protein coupled receptors and protein kinases).